Lab 3 (Isolation of RNA and Analysis of Gene Expression by RT-PCR)

Question 1

What are the objectives of this lab?

Answer 1

1) Isolate total RNA from cultured human cells.

2) Convert mRNA into cDNA using reverse transcription.

3) Analyse gene expression using endpoint and real-time PCR (qPCR).

Question 2

Background for this Lab

Answer 2

Lab 3 focuses on studying gene expression by analysing mRNA levels. Traditionally, techniques like Northern blotting and RNase protection assays were employed, which were time-consuming and required radioactive probes. However, advancements in real-time PCR technology allow for a more sensitive and rapid analysis by combining reverse transcription with PCR (RT-PCR).

Before PCR can be used to analyse mRNA levels, the RNA must first be converted into cDNA using the enzyme reverse transcriptase (RT). This enzyme requires a primer to start the process, often an oligonucleotide composed of 18-20 deoxythymidines (oligo dT).

The oligo dT binds to the poly(A) tails of mRNA molecules, and the reverse transcriptase then synthesizes a complementary DNA strand (cDNA) based on the mRNA template. This process is referred to as first-strand cDNA synthesis.

Question 3

Summary of this Experiment (Week 1 and 2)

Answer 3

In Lab 3, you will purify RNA from cultured human HCT116 colon cancer cells and compare gene expression patterns between cells with and without functional p53. The cells are treated with the chemotherapy agent 5-fluorouracil (5-FU) or left untreated. The experiment aims to measure the effect of p53 on the induction of the p21 gene in response to 5-FU treatment.

1) Week 1: Isolate and purify total RNA from two types of colorectal cancer cells, both treated and untreated. Quantify the RNA spectrophotometrically and assess its quality on an agarose gel. Perform first-strand cDNA synthesis using reverse transcriptase.

2) Week 2: Perform endpoint and real-time PCR analysis on the cDNA to analyse p21 expression, comparing it to the expression of the housekeeping gene GAPDH.

Question 4

How is RNA Isolated?

Answer 4

RNA is more prone to degradation than DNA due to its chemical structure and the presence of ubiquitous RNases. Therefore, careful handling and specific techniques are crucial for successful RNA isolation.

Guanidinium thiocyanate-phenol-chloroform extraction is a common method used to purify RNA. This method utilizes guanidinium thiocyanate, a chaotropic agent, which denatures proteins, including RNases, protecting RNA from degradation. Phenol and chloroform are organic solvents used to separate the RNA from DNA and proteins. The RNA remains in the aqueous phase, while DNA and proteins partition into the organic phase.

Question 5

RNase-free Water

Answer 5

Used throughout the experiment to prevent RNase contamination and degradation of RNA.

Question 6

Lysis Solution

Answer 6

Contains guanidinium thiocyanate to lyse cells and denature RNases, protecting RNA.

Question 7

Chloroform

Answer 7

An organic solvent used to separate RNA from DNA and proteins during extraction.

Question 8

Isopropanol

Answer 8

Precipitates RNA from the aqueous phase

Question 9

70% Ethanol

Answer 9

Washes the RNA pellet to remove residual salts and contaminants.

Question 10

TBE Buffer

Answer 10

Used for agarose gel electrophoresis to seperate and visualise RNA.

Question 11

RNA Loading Dye

Answer 11

Contains formamide to denature RNA and tracking dyes to visualise RNA migration during electrophoresis.

Question 12

Oligo (dT)19 Primer

Answer 12

Binds to the poly(A) tail of mRNA, providing a starting point for reverse transcriptase.

Question 13

dNTP Mix

Answer 13

Building blocks (dATP, dCTP, dGTP, dTTP) for cDNA synthesis.

Question 14

MuLV Reverse Transcriptase

Answer 14

An RNA-dependent DNA polymerase that synthesises cDNA from the mRNA template.

Question 15

5X RT Reaction Buffer

Answer 15

Provides optimal conditions (pH, salts) for reverse transcriptase activity.

Question 16

PCR Buffer

Answer 16

Provides optimal pH and salt conditions for DNA polymerase activity during PCR.

Question 17

Forward and Reserve Primers

Answer 17

Short, single-stranded DNA sequences that are specific to the target genes (GAPDH and p21) and are used to amplify the cDNA during PCR.

Question 18

Taq DNA Polymerase

Answer 18

A thermostable DNA polymerase that amplifies DNA during PCR.

Question 19

MgCL2

Answer 19

Acts as a cofactor for DNA polymerase.

Question 20

2X qPCR Mix

Answer 20

Contains a thermostable DNA polymerase, dNTPs, and a fluorescent dye (SYBR Green I) for qPCR.

Question 21

SYBR Green I Dye

Answer 21

A fluorescent dye that binds to double-stranded DNA and is used to detect and quantify PCR products in real-time.

Question 22

DNA Ladder

Answer 22

A set of DNA fragments of known sizes, used as a reference to determine the size of the PCR products on an agarose gel.

Question 23

Why is RNA more susceptible to degradation than DNA? How can RNA degradation be prevented during isolation?

Answer 23

RNA is more prone to degradation than DNA due to the presence of a 2’-hydroxyl group on the ribose sugar, which makes it chemically more reactive. Also, RNases, enzymes that degrade RNA, are ubiquitous and highly stable. To prevent RNA degradation during isolation, the following precautions are taken:

1) Use RNase-free reagents and equipment: All reagents and materials used should be certified RNase-free.

2) Work quickly and on ice: Keeping the samples cold slows down enzymatic activity.

3) Use a chaotropic agent: Guanidinium thiocyanate in the lysis solution denatures RNases, rendering them inactive.

4) Wear gloves: Gloves prevent RNase contamination from hands.

Question 24

What is the purpose of reverse transcription in this experiment? Describe the steps involved in first-strand cDNA synthesis.

Answer 24

Reverse transcription is necessary to convert mRNA into cDNA, which is more stable and suitable for PCR amplification. The steps involved in first-strand cDNA synthesis are:

1) Annealing: The oligo (dT)18 primer anneals to the poly(A) tail of the mRNA.

2) Extension: Reverse transcriptase uses the mRNA as a template and the dNTPs as building blocks to synthesize a complementary DNA strand (cDNA).

3) Inactivation: The reaction is heated to inactivate the reverse transcriptase enzyme.

Question 25

Why is it important to include a no RT control in this experiment?

Answer 25

The no RT control is essential to ensure that any PCR products detected are derived from cDNA synthesized from RNA and not from contaminating genomic DNA. The no RT control lacks reverse transcriptase; therefore, no cDNA should be synthesized. If a PCR product is observed in the no RT control, it indicates the presence of DNA contamination.

Question 26

Explain the difference between endpoint PCR and qPCR. What information can be obtained from each method?

Answer 26

(1) Endpoint PCR:
- Traditional PCR method where the amplified product is detected at the end of the reaction.
- Amplified products are visualised on an agarose gel, allowing for qualitative analysis of gene expression (presence or absence of a PCR product) and size determination of the PCR product.
- Semi-quantitative: Band intensity can provide a rough estimate of relative gene expression levels.

(2) qPCR:
- Also known as real-time PCR, it allows for the quantification of PCR product accumulation during each cycle of the reaction.
- Utilises fluorescent dyes, such as SYBR Green I, that bind to double-stranded DNA. The fluorescence signal increases proportionally to the amount of PCR product generated.
- Quantitative: Provides precise measurements of gene expression levels by determining the cycle threshold (Ct) value, which is the cycle number at which the fluorescence signal crosses a predefined threshold.

Question 27

What is a housekeeping gene, and why is it important to analyse its expression in this experiment?

Answer 27

• Housekeeping genes are genes that are constitutively expressed at relatively constant levels in different cell types and under different experimental conditions. In this experiment, GAPDH is used as a housekeeping gene.
• Analysing the expression of a housekeeping gene serves as an internal control to:

(1) Normalise the expression of the target gene (p21): This accounts for variations in RNA extraction efficiency, reverse transcription efficiency, and sample loading.

(2) Ensure that observed changes in p21 expression are not due to general changes in gene expression.
By comparing the expression of p21 to that of GAPDH, it is possible to determine whether changes in p21 expression are specific to the experimental conditions (p53 status and 5-FU treatment).

Question 28

Based on your understanding of p53 and p21, what results would you expect to see in the qPCR analysis of p21 expression in wild-type and p53-null cells treated with 5-FU?

Answer 28

• p53 is a tumor suppressor gene that is activated in response to DNA damage, such as that induced by 5-FU. p53, in turn, activates the transcription of p21, a cell cycle inhibitor. Therefore, we would expect the following results:

(1) Wild-type cells treated with 5-FU: Increased p21 expression compared to untreated wild-type cells. This is because functional p53 will induce p21 transcription in response to DNA damage.

(2) p53-null cells treated with 5-FU: No significant change in p21 expression compared to untreated p53-null cells. This is because the absence of functional p53 prevents the induction of p21 transcription, even in the presence of DNA damage.

These results would confirm the role of p53 in regulating p21 expression in response to DNA damage and highlight the importance of p53 in tumor suppression.